Cytokine receptors and receptor tyrosine kinases (RTKs) are large integral membrane proteins with single membrane-spanning helices. Representative high-resolution structures have been determined of the ligand binding domains for both classes of receptor, and of the intracellular kinase domain for the RTKs. However, structures of the transmembrane (TM) and juxtamembrane (JM) regions of these receptors are generally lacking. An emerging view of both receptor families is that pre-formed dimers with high-affinity binding sites are the physiologically important forms of the receptors. In these receptors, the intracellular domains are in close proximity, but remain inactive until ligand binding triggers a conformational change in the receptor dimer. A common element of both receptor families is that the intracellular JM region plays a key regulatory role. We hypothesize that a change in the orientation of the TM helices releases inhibitory constraints within the JM region allowing the receptor to switch from an inactive to an active conformation. We propose to show how the rotational orientation of the TM helices is coupled to changes in the relative orientation and/or structure of the JM regions of these biologically important single helix membrane proteins. A combination of biophysical methods will be used, including solid-state and solution NMR, fluorescence and infrared spectroscopy. The proposed studies on TM helix interactions are part of a long-range effort to understand how membrane proteins fold and function. The proposed studies on the JM regions of these receptors address questions involving the role of membrane lipids, such as the phosphoinositides, in regulating receptor activity. The significance of the proposed research is to provide a molecular basis for disease. Mutations and deletions in the TM and JM domains of the RTKs have been identified in a variety of human tumors that result in constitutive receptor activity. Mutations in the TM and JM domains of the Epo and Tpo receptors produce erythroleukemia and myeloproliferative disorders, respectively. The four specific aims of the grant are to determine the structure and function of the TM and JM regions of these single TM helix receptors in order to have a comprehensive view of the full receptor structure. PUBLIC HEALTH RELEVANCE: Membrane receptors are involved in most cellular processes and are the target of the majority of pharmaceuticals currently on the market. The aim of the research is to establish how receptors in the receptor tyrosine kinase and cytokine receptor families with a single transmembrane sequence transmit information across cell membranes. We propose to determine the structures of the membrane-spanning region of these receptors in their active and inactive conformations, and to establish the structural changes that result from disease-causing mutations.